Unit V - Microbiology

Concept of Single celled Organisms

Life on earth evolved from a single cell millions of years ago. These single cells are known as unicellular
organisms. All the life processes in a unicellular organism, including digestion, excretion, respiration, occur
within a single cell. These cannot be seen by naked eyes and are hence called microorganisms. There are several
kinds of unicellular organisms such as bacteria, protozoa, algae, fungi, etc.
Characteristics of Unicellular Organisms
The characteristics of unicellular organisms are as follows:

1. The unicellular organisms usually reproduce by asexual means.

2. They can be eukaryotes or prokaryotes.
3. They are found in almost all habitats, from hot springs to frozen tundra.
4. They possess whip-like structures for movement.
5. The nutrients enter or leave the cell by the process of diffusion.

Types of Unicellular Organisms

There are two types of unicellular organisms:

 Prokaryotes
 Eukaryotes
Prokaryotes

 Prokaryotes are unicellular organisms without a true nucleus.

 They are very small in size from 0.1 to 5.0 µm. This facilitates the diffusion of ions and molecules to
different parts of the cell.
 They have a peptidoglycan cell wall.
 The cell wall helps to maintain the shape of the cell and prevents dehydration.
 They use flagella for locomotion.
 They possess fimbriae for attachment to the host cell, and pili to exchange genetic material during
conjugation.
 Archaebacteria, eubacteria are a few examples of prokaryotes.
Eukaryotes

 Eukaryotes are larger than prokaryotes, with a membrane-bound nucleus.

 The DNA is present in the nucleus of the cell.
 Eukaryotes can be plant cells or animal cells.
 The plant cells contain vacuoles, chloroplast and a large central vacuole. These cannot be found in
animal cells.
 Lysosomes and centrosomes are present in animal cells and not in plant cells.
 Eukaryotes include Protozoa and Protista.
Nutrition in Unicellular Organisms
Unicellular organisms feed on other organisms or liquid matter. The digestion is intracellular. Larger particles
are ingested by phagocytosis or pinocytosis. The smaller sized particles enter into the cell through osmosis and
diffusion.
Reproduction in Unicellular Organisms
 Unicellular organisms reproduce by the following ways:

 The unicellular organisms reproduce by binary fission. In this, a single cell divides, giving rise to two
daughter cells. This can be seen in bacteria and amoeba.
 The yeast cells reproduce by the process called budding.
 Sometimes amoeba reproduces by the process called encysting. During unfavourable conditions, it
forms a protective covering around itself called a cyst. This cyst contains chitin that helps it to
reproduce.
Respiration in Unicellular Organisms
The unicellular organisms respire aerobically or anaerobically. They do not have any respiratory structures for
respiration. They respire by the process of diffusion through the skin surface.
Unicellular Organisms Examples
Following are some of the examples of unicellular organisms:

 Escherichia coli
 Diatoms
 Protozoa
 Protista
 Streptococcus
 Pneumococci
 Dinoflagellates

Classification of Microorganisms

Microorganisms are categorized into four major groups:

 Bacteria: These are microscopic, single-celled organisms that grow in diverse environments. These organisms
can live in soil, the ocean, and also in the human gut. These act in a positive way by participating in curdling
milk into yoghurt and helping in digestion.

 Fungi: These are a group of microorganisms that are eukaryotic in nature and these consist of microorganisms
such as yeasts, moulds and mushrooms. These are grouped into a kingdom and are different from other
eukaryotic life kingdoms of plants and animals.

 Algae: These are simple, non-flowering, and typically aquatic plants of a huge group that have seaweeds and
many single-celled forms included in them. Algae has chlorophyll in it but lacks true stems, roots, leaves, and
vascular tissue. Algology or Phycology is the study of algae and it has a range of photosynthetic organisms and
many are not closely related. They are a polyphyletic group.

 Protozoa: Protozoa (also protozoan, plural protozoans) is an informal term for single-celled eukaryotes, either
free-living or parasitic, which feed on organic matter such as other microorganisms or organic tissues and
debris.

Apart from the above microorganisms, viruses are microscopic but differ in their reproduction aspects as these
reproduce only in cells of their hosts. The host organisms are animals, bacteria, or plants.

 Viruses: Viruses usually consist of little more than a strand of DNA or RNA enclosed in a simple
protein shell known as a capsid. Sometimes the complete nucleocapsid may be enclosed in a lipoprotein
 envelope derived largely from the host cell. Viruses are capable of growing only within the living cells
of an appropriate animal, plant or bacterial host; none can grow in an inanimate nutrient medium. The
viruses that infect and parasitize bacteria are termed bacteriophages or phages.

 Mycoplasmas: Mycoplasmas are the smallest and simplest self-replicating bacteria. The mycoplasma
cell contains the minimum set of organelles essential for growth and replication: a plasma membrane,
ribosomes, and a genome consisting of a double-stranded circular DNA molecule. Unlike all other
prokaryotes, the mycoplasmas have no cell walls, and they are consequently placed in a separate
class Mollicutes (mollis, soft; cutis, skin). The trivial term mollicutes is frequently used as a general term
to describe any member of the class, replacing in this respect the older term mycoplasmas.

Ecological aspects of micro-organisms

 It is the relationship of microorganisms with one another and with their environment. Microorganism
impacts the entire biosphere. They are present virtually in all part of planet earth including some in extreme
conditions such as acidic lakes to deep ocean and from frozen environment to hydrothermal vents.
 Environmental microbiology includes microbial biodegradation of domestic, agricultural and industrial
wastes and also sub-surface pollution in soil, sediments and marine environments.

Role of microorganism in ecosystem –

Generate Oxygen in atmosphere:

The other area in which microorganisms play their part is the production of oxygen. The rainforests are
considered to be the lungs of the world but still they provide half of the oxygen, the other half is provided by the
microorganisms. It is a fact that microorganisms are the source of producing oxygen in the environment. Single
celled green plants especially include in this category of microorganisms like cyanobacteria and blue green
algae. The habitat of these plants is the oceans, lakes and ponds. It is not that these plants only produce oxygen
but they also produce food that is why they are called as autotrophs.

Recycle nutrition stored in organic matters to inorganic form:

Microbial decomposition of organic matters releases the minerals such as N, P, K etc and these minerals are
bound up and made available for producers (green plants) to use. Without this recycling primary productivity of
ecosystem would stop. In soil, fungi are the most important decomposers of plant materials and are followed by
bacteria.

Fix atmospheric Nitrogen into usable form:

The only organisms capable of fixing atmospheric nitrogen into useable form are bacteria. These nitrogen fixing
bacteria are Rhizobium, Cyanobacteria etc

Microorganisms give plant roots access to nutrition in soil:

Plant root creates a zone of nutrition depletion around their surface by using available nutrition. A group of
fungi called mycorrhiza found associated with root hairs of plants helps in effective absorption of nutrition in
soil.

Decompositions:
Microorganisms are the main source of decomposition. When an animal or human die, they penetrate in their
bodies and deep inside, they start digesting the dead skin and other organs. When the microorganisms
decompose a body, in that process many useful nutrients are released from the body which help in many
processes. If there were no microorganisms then it would be impossible for the dead body to decompose and
nutrients would have lost somewhere. Due to the process of decomposition, the soil becomes fertile and can be
beneficial for the production of plants. The soil cannot be fertilized without microorganisms.

Parasitic nature of microorganisms:

Though it seems negative property of the microorganisms that they cause diseases in the living organisms due
to the parasitic characteristics in them, but if we see from other aspect, they are the cause of bringing evolution
of many species because weak organisms die out due to the disease and strong and beneficent organisms are left
out in the ecosystem. It is their parasitic nature which kills the weak individuals from the community and leaves
enough food for those who survive so that they can grow and reproduce efficiently and healthily.

Symbiotic properties:
One more important role of the microorganisms is that they create symbiotic relationship with higher organisms
like cows and camels and sheep etc. when cow eats grass, it cannot digest it independently but there are certain
microbes involved which help it to digest the food. Other benefit point is that when termites eat wood, they are
not able to digest it but there is a special microorganism in their gut which helps to digest wood. When humans
are concerned, they are also not able to digest their food without microbes. There is bacterial specie in our
intestine E.coli which helps to digest the food properly.

Concept of species and strains

Species concept:

There is a wide range of approaches to define how we identify species and how species function in nature, each
approach is known as species concept. The number and types of species concepts which exist are constantly
changing from time to time. Here are 4 main types of species concept which are as follows:-

1. Biological species concept

2. Morphological species concept
3. Ecological species concept
4. Phylogenetic species concept

1. Biological species concept:

- It is the most widely accepted species concept. It defines species in terms of interbreeding.
- It states that species are groups of interbreeding natural populations that are reproductively isolated from
other such groups and can produce viable offspring.

2. Morphological (Phenetic) species concept:

- It defines species as a group of organisms sharing a unique combination of physical characteristics.
- A species may be morphologically diverse (eg. Dogs) or distinct species may be morphologically
indistinguishable (eg. Cryptic species).

3. Ecological species concept:

 - According to the ecological species concept the more similar two organisms are more likely to overlap
the need and will compete over resources such as food and shelter.
- That is they share the exact same ecological niche.

4. Phylogenetic species concept:

- It defines a species as a group of organisms that shares a common ancestor and can be distinguished
from other organisms that do not share that ancestor.
- Phylogenetic species concept asserts that on the tree of life, species are the distal twigs.

Strains:

- A strain is a subset of a bacterial species i.e. it is a low-level taxonomic rank, usually at intraspecific
level (within a group) differing from other bacteria of the same species by some minor but identifiable
difference and different functions.
- Strains refer to the small changes in bacterial genotypes. Genotypes list only genes that are mutated in
the bacterial strains.

Microbial Growth kinetics

Bacteria are prokaryotic organisms that most commonly replicate by the asexual process of binary fission.
These microbes reproduce rapidly at an exponential rate under favorable conditions. When grown in culture, a
predictable pattern of growth in a bacterial population occurs. This pattern can be graphically represented as the
number of living cells in a population over time and is known as a bacterial growth curve. Bacterial growth
cycles in a growth curve consist of four phases: lag, exponential (log), stationary, and death.

Bacteria require certain conditions for growth, and these conditions are not the same for all bacteria. Factors
such as oxygen, pH, temperature, and light influence microbial growth. Additional factors include osmotic
pressure, atmospheric pressure, and moisture availability. A bacterial population's generation time, or time it
takes for a population to double, varies between species and depends on how well growth requirements are met.

In nature, bacteria do not experience perfect environmental conditions for growth. As such, the species that
populate an environment change over time. In a laboratory, however, optimal conditions can be met by growing
bacteria in a closed culture environment. It is under these conditions that the curve pattern of bacterial growth
can be observed.

The bacterial growth curve represents the number of live cells in a bacterial population over a period of time.
  Lag Phase: This initial phase is characterized by cellular activity but not growth. A small group
of cells are placed in a nutrient rich medium that allows them to synthesize proteins and other molecules
necessary for replication. These cells increase in size, but no cell division occurs in the phase.
 Exponential (Log) Phase: After the lag phase, bacterial cells enter the exponential or log phase. This is
the time when the cells are dividing by binary fission and doubling in numbers after each generation
time. Metabolic activity is high as DNA, RNA, cell wall components, and other substances necessary for
growth are generated for division. It is in this growth phase that antibiotics and disinfectants are most
effective as these substances typically target bacteria cell walls or the protein synthesis processes
of DNA transcription and RNA translation.
 Stationary Phase: Eventually, the population growth experienced in the log phase begins to decline as
the available nutrients become depleted and waste products start to accumulate. Bacterial cell growth
reaches a plateau, or stationary phase, where the number of dividing cells equal the number of dying
cells. This results in no overall population growth. Under the less favorable conditions, competition for
nutrients increases and the cells become less metabolically active. Spore forming bacteria produce
endospores in this phase and pathogenic bacteria begin to generate substances (virulence factors) that
help them survive harsh conditions and consequently cause disease.
 Death Phase: As nutrients become less available and waste products increase, the number of dying cells
continues to rise. In the death phase, the number of living cells decreases exponentially and population
growth experiences a sharp decline. As dying cells lyse or break open, they spill their contents into the
environment making these nutrients available to other bacteria. This helps spore producing bacteria to
survive long enough for spore production. Spores are able to survive the harsh conditions of the death
phase and become growing bacteria when placed in an environment that supports life.

Sterilization and media compositions

The food material or substances required for growing microorganisms in vitro (outside the body) is called
culture medium.

Media:

- Bacteria and fungi are grown on or in microbiological media of various types.

- Different nutrients may be added to the medium, making it higher in protein or in sugar. Various pH
indicators are often added.
- In this exercise, you will make all purpose media called nutrient broth (liquid) and nutrient agar (solid)
are the exact same formula save for the addition of agar-agar, an extract from the cell walls of red algae.
- The most common growth media nutrient broths or LB medium are liquid.
- These are often mixed with agar and poured into Petri dishes to solidify.
- These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as
very few bacteria are able to decompose agar. Many microbes can also be grown in liquid cultures
comprised of liquid nutrient media without agar.

Composition of culture media:

- Water
- Energy source
- Carbon source
- Nitrogen source
- Mineral salts
- Special growth factors
Types of media:

- A defined medium will have known quantities of all ingredients like, trace elements, vitamins, carbon
and nitrogen source. Glucose or glycerols are often used as carbon sources, and ammonium salts or
nitrates as inorganic nitrogen sources.
- An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture
of many, many chemical species in unknown proportions.

Based on ingredients, medium classified as various types:

1. Nutrient media – a source of amino acids and nitrogen (eg. Beef, yeast extracts). This is an
undefined medium because the amino acid source contains a variety of compounds with the
exact composition being unknown. These media contain all the elements that most bacteria need
for growth and are non-selective, so they are used for the general cultivation and maintenance of
bacteria kept in laboratory culture collections.
2. Minimal media – media that contains the minimum nutrients possible for colony growth,
generally without the presence of amino-acids, and are often used by microbiologists and
geneticists to grow “wild type” microorganisms. These media can also be used to select for or
against the growth of specific microbes. Usually a fair amount of information must be known
about the microbe to determine its minimal media requirements.
3. Selective media – used for the growth of only selective microorganisms. For example, if a
microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that
antibiotic can be added to the medium in order to prevent other cells, which do not possess the
resistance from growing.
4. Differential media – also known as indicator media are used to distinguish one microorganism
type from another growing on the same media. This type of media uses the biochemical
characteristics of a microorganism growing in the presence of specific nutrients or indicators
(such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly
indicate the defining characteristics of a microorganism. This type of media is used for the
detection and identification of microorganisms.

Based on physical state, medium classified as 3 types –

1. Solid medium – contains solidifying agents (agar)

2. Liquid medium – without agar (broth) & producing turbidity (growth)
3. Semi solid medium – demonstrating bacterial motility

Sterilization technique:

 When microbiological media has been made, it still has to be sterilized because of microbial
contamination from air, glassware, hands, etc. Within a few hours there will be thousands of bacteria
reproducing in the media so it has to be sterilized quickly before the microbes start using the nutrients
up.
 The sterilization process is a 100% kill, and guarantees that the medium will stay sterile. Media
sterilization is carried out with the autoclave, basically a huge steam cooker. Steam enters into a jacket
surrounding the chamber. When the pressure from the steam is at a certain point in the jacket, a valve
allows the steam to enter the chamber.
 The pressure will go up over 15 pounds per square inch (psi): at this point the timer begins to count
down – usually for 15 minutes, depending on the type of media. The high pressure in a closed container
 allows the temperature to go above the highest temperature one could get by just boiling, around 121̊ C.
Therefore, the parameters for sterilization with an autoclave are 121̊ C at˃15 psi for 15 minutes.

Methods of sterilization:

Physical method –

1. Moist heat – autoclave and pressure cooker

2. Dry heat – hot air oven
3. Radiation – UV radiation
4. Sterilization by membrane filtration – Millipore membrane filter

Chemical methods –

1. Alcohol – ethanol and isopropanol (70%)

2. Aldehyde – formaldehyde
3. Inorganic chemicals – heavy metals; copper, mercury, etc.

Microscopy

The definition of a microscope is an optical instrument used for viewing very small objects. These small
objects can be anything from cells and microscopic life to the molecular structure of objects. The term for the
act of using a microscope is called microscopy, and there are several different types of microscopy that are
differentiated by the type of microscope used. The term "microscope" comes from the Greek words for "small"
and "to view", thus a microscope is a tool to view small things with.

Different Types of Microscopes

There are many different types of microscopes, each with a different use. The use of these different types of
microscopes makes up the different categories of microscopy. There are three main types of microscopy, each
defined by the type of microscope used in the work. The three types of microscopy are:-
1. Optical microscopy which uses optical microscopes
2. scanning probe microscopy which uses scanning probe microscopes, and
3. electron microscopy which uses electron microscopes.

Parts of Microscope:

The body is made from metal and the parts are divided into mechanical and ocular parts.
1. Base: The bottom part is called as base. It gives support to the body. The illuminating source of microscope is
placed at the base of microscope
2. Arm: It connects the ocular part with the base. The arm is used to hold and carry the microscope. The coarse
and fine focusing knobs are placed on the arm.
3. Eyepiece: It is present at the top of microscope. It is the part through which we observe the sample/object. The
magnifying power of the eyepiece lens is generally 10X.
4. Eyepiece tube or body tube: The function of eyepiece tube is to hold the eyepiece and hence it is names as
eyepiece tube.
5. Nosepiece: This part connects the eyepiece tube to objective lenses. The flexibility of nosepiece allows
switching the objective lenses.
6. Objective lens: In compound microscope, in general 3 objective lenses are placed. Their magnifying power is
10X, 40X and 100X respectively. The total magnification of respective lenses would be 10X x 10X = 100X,
40X x 10X = 400X and 100X x 10X = 1000X (the magnifying power of eyepiece lens is 10X).
7. Focusing mechanism (Adjacent knobs): The coarse and fine adjustment knobs are used for right placing of
sample and for focusing it.
8. Stage: It is place where sample or object is kept for viewing. For accurate placement, the stage is provided with
clips that hold the slide firmly. The adjustment knobs are used to move the clips as per requirement.
9. Aperture: It is present on the stage that allows the entry of light from illuminator to fall on sample or object.
10. Source of Illumination: In non-electric microscope, the sunlight is used as source of illumination and hence
mirror is placed to focus the sunlight. In electric microscope, lamp is placed of specific wavelength.
11. Condenser: It is placed at the bottom of the stage. Its function is to focus the light on sample form illuminator.
The lens quality of condenser equally affects the superiority of image.
12. Diaphragm: It is generally associated with condenser. And it is also placed at the bottom of the stage, beside
condenser. The diaphragm and condenser together produce hollow cone of light that strikes the sample and
illuminate it.